C. dubliniensis and C. tropicalis fail to strongly upregulate ECE1 expression in vitro or in vivo. (A) Overnight YPD cultures of C. albicans, C. dubliniensis, and C. tropicalis were transferred to either fresh YPD or RPMI medium for 4 h. RNA was extracted, normalized, and reverse transcribed, and the expression of ECE1 and the housekeeping gene ACT1 was monitored. The normalized fold expression of ECE1 (mean plus SEM) was calculated from the average independent biological repeats (n = 3) using the ΔΔCT method. Statistical significance was calculated using one-way ANOVA and Dunnett’s posttest. *, P < 0.05; ***, P < 0.001. (B) C. albicans, C. dubliniensis, and C. tropicalis were intravaginally inoculated in estrogen-treated mice as described in the text. At day 3 p.i., the mice underwent two rounds of vaginal lavage with PBS to recover fungal cells, and RNA was immediately prepared as described above. The normalized fold expression of ECE1 (mean plus SEM) was calculated from the average of independently inoculated animals (n = 4) and compared to ACT1 expression using the ΔCT method. n.d., not detected.